Rapamycin is a neuroprotective treatment for traumatic brain injury

Abstract

The mammalian target of rapamycin, commonly known as mTOR, is a serine/threonine kinase that regulates translation and cell division. mTOR integrates input from multiple upstream signals, including growth factors and nutrients to regulate protein synthesis. Inhibition of mTOR leads to cell cycle arrest, inhibition of cell proliferation, immunosuppression and induction of autophagy. Autophagy, a bulk degradation of sub-cellular constituents, is a process that keeps the balance between protein synthesis and protein degradation and is induced upon amino acids deprivation. Rapamycin, mTOR signaling inhibitor, mimics amino acid and, to some extent, growth factor deprivation. In the present study we examined the effect of rapamycin, on the outcome of mice after brain injury. Our results demonstrate that rapamycin injection 4 h following closed head injury significantly improved functional recovery as manifested by changes in the Neurological Severity Score, a neurobehavioral testing. To verify the activity of the injected rapamycin, we demonstrated that it inhibits p70S6K phosphorylation, reduces microglia/macrophages activation and increases the number of surviving neurons at the site of injury. We therefore suggest that rapamycin is neuroprotective following traumatic brain injury and as a drug used in the clinic for other indications, we propose that further studies on rapamycin should be conducted in order to consider it as a novel therapy for traumatic brain injury.

Introduction

The mammalian target of rapamycin (mTOR) is a phosphatidylinositol kinase-related serine–threonine kinase (Schmelzle and Hall, 2000). Growth factors, mitogens and hormones activate the PI3K/Akt signaling pathway and consequently the mTOR signaling (Hay and Sonenberg, 2004). Nutrients (amino acids, glucose) also regulate mTOR activity (Proud, 2004). Thus, mTOR functions by integrating extracellular signals (growth factors and hormones), with amino acid accessibility and intracellular energy status to control translation rates and additional metabolic processes (Hay and Sonenberg, 2004). mTOR regulates a wide array of cellular functions, including translation, transcription, mRNA turnover, protein stability, actin cytoskeletal organization and autophagy (Harris and Lawrence, 2003). The best-characterized function of mTOR in mammalian cells is regulation of translation. Ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E binding protein 1 (4EBP1), the most extensively studied substrates of mTOR, are key regulators of protein translation (Harris and Lawrence, 2003). mTOR regulates eIF4G, S6K, 4EBP1 and Atg1 proteins by phosphorylation. As a consequence, mTOR enhances translation initiation and affects cell growth and proliferation. Growth factors, such as insulin, and nutrients, such as amino acids or glucose, enhance mTOR function, as evidenced by an increased phosphorylation of S6K and 4EBP1 (Harris and Lawrence, 2003).

Rapamycin is a macrolide antibiotic first developed as an antifungal agent, however, it was discovered that rapamycin had potent immunosuppressive and antiproliferative properties. Rapamycin binds to the cytosolic protein FK-binding protein 12 (FKBP12). Thereby, the rapamycin–FKBP12 complex can inhibit mTOR preventing further phosphorylation of P70S6K, 4EBP1 and, indirectly, other proteins involved in transcription and translation and cell cycle control (Vignot et al., 2005). Inhibition of mTOR leads to, among others, cell cycle arrest in tumor cells resulting in growth retardation. Antiapoptotic signals mediated by mTOR are also antagonized by rapamycin (Guba et al., 2002). Rapamycin has been shown to inhibit growth of melanoma cells in mouse model, additionally, as an immunosuppressor it prevents transplant rejection in organ transplant recipients (Koehl et al., 2004). Apart from its immunosuppressive capacity, rapamycin was also recently shown to be capable of preventing coronary artery re-stenosis (Sousa et al., 2003).

The observation that rapamycin treatment also induces autophagy indicates that mTOR kinase activity is involved in this process (Kamada et al., 2004). Autophagy is a process of bulk degradation of cellular constituents through an autophagosomic–lysosomal pathway (Klionsky and Emr, 2000, Wang and Klionsky, 2003). Autophagy is important in normal growth control and may be defective in diseases (Larsen and Sulzer, 2002, Ogier-Denis and Codogno, 2003). This process permits the disposal of unwanted damaged organelles in the cell and allows recycling of free amino acids and nutrients at time of nutrient deprivation or other insults. Thus, autophagy is important for normal cell growth, differentiation and survival (Reggiori and Klionsky, 2002). Recent studies demonstrated that enhancement of autophagy may induce degradation of unwanted aggregates such as of mutant huntingtin, indicating that at least in some neurodegenerative diseases enhancement of autophagy may rescue neuronal cells (Ravikumar and Rubinsztein, 2004). It was previously shown that rapamycin can protect against mutant huntingtin-induced degeneration in cells, fly and mouse models of Huntington’s disease (Ravikumar et al., 2002, Ravikumar et al., 2004). It was also demonstrated that induction of autophagy by rapamycin enhances the clearance of a wide range of aggregate-prone proteins and reduces their toxicity (Berger et al., 2006, Webb et al., 2003). The mammalian target of rapamycin (mTOR) negatively regulates autophagy (Schmelzle and Hall, 2000). Atg1–Atg13 complex is regulated by mTOR, which is also involved in the regulation of transcription, translation and cell cycle and plays a central role in cell metabolism. Thus, inhibition of mTOR may induce autophagy (Klionsky and Emr, 2000, Wang and Klionsky, 2003), may result in a reduction of protein synthesis and may inhibit cell proliferation (Dutcher, 2004).

Brain damage following closed head injury is divided to primary and secondary injuries. While primary brain injury results from the mechanical forces applied to skull and brain at the time of injury, the secondary neuronal injury is associated with a neuroinflammatory response characterized by microglial and astrocytic activation, resulting in the release of reactive oxygen species and inflammatory cytokines (Leker and Shohami, 2002). Several studies described the loss of neuronal cells that follows injury as both necrotic and apoptotic (Faden, 1993). Recently we have demonstrated that following closed head injury (CHI) or stab injury in mice the levels of Beclin 1, used as a marker for autophagy, are elevated near the injury site in neurons and astrocytes (Diskin et al., 2005). These findings suggest that Beclin 1 and autophagy may play a role in brain responses to head trauma.

In the present study we examined the effect of rapamycin treatment on recovery from traumatic brain injury (TBI). Our results do not exclude the existence of other mechanism except autophagy in response to rapamycin treatment. Beyond doubt, our results indicate that rapamycin treatment significantly improved the recovery from head injury and increased the number of surviving neurons at the injury site. Thus rapamycin treatment should be evaluated for patients suffering from traumatic brain injury.